It is known that glutaminase plays an important role in food industry, particularly in enzymatically degradating protein to produce flavoring foods.
For enzymatically degradating protein, the protein is degradated finally into its constitutional amino acids via peptides by action of various proteolytic enzymes.
Glutamine formed in such cases is readily converted non-enzymatically into tasteless pyroglutamic acid in the absence of glutaminase.
The rate of degradating glutamic acid into pyroglutamic acid is increased with an increasing temperature.
It is known that the coexistence of glutaminase in such a degradation system enables not only prevention of conversion of glutamine into pyroglutamic acid but also conversion of free glutamine into its corresponding amount of glutamic acid.
Enzymatic degradation of protein at high temperatures is a very effective method for raising the rate of degradation of the protein and preventing contamination with microorganisms, thus efficiently producing glutamic acid-rich flavoring foods.
To adopt these conditions, however, it is necessary that usable glutaminase is thermostable.
Further, a method of making soy sauce, miso etc. by fermentation is known as a process for producing glutamic acid-rich flavoring foods by enzymatically degradating protein materials in the presence of high conc. salt. In this case, the presence of high conc. salt is intended to prevent decomposition and putrefaction resulting from microbial contamination.
However, it is known that a lower glutamic acid content after degradation is an inevitable disadvantage in the enzymatic degradation method and this glutamic acid content is considerably lower than in a hydrochloric acid degradation method.
The speculative reason for the lower glutamic acid content in the enzymatic degradation method is that glutaminase is significantly inhibited by high conc. salt in soy sauce flavorings obtained by admixing soy sauce koji with saline, so glutamine released from protein materials by enzyme degradation is rapidly converted into pyroglutamic acid.
For this reason, it is also proposed that separately prepared glutaminase is added to soy sauce flavorings to convert them into glutamic acids. However, because the glutaminase added in this method is not resistant to salt, salt is initially mixed at a concentration as low as about 7 to 8%, and the glutaminase is then allowed to act for a predetermined period of time, followed by supplementing the materials with salt to an usual salt concentration of 15% or more for flavorings.
However, microbial contamination cannot be prevented in the presence of salt at such low concentration, so there is the risk of deterioration in qualities of the product.
The problem with simultaneous use of this kind of glutaminase is that the glutaminase used is not resistant to salt, and that the procedures of initially adding low concentration of salt and then raising the concentration of salt are cumbersome.
Accordingly, it is important to obtain a glutaminase capable of acting adequately even in the presence of high conc. of salt.
Candida famata (Japanese Patent Publication No. 38748/1994) and Cryptococcus albidus (Japanese Patent Publication No. 48759/1974) are known respectively to produce glutaminases having salt resistance and thermostability.
However, the relative activity of the former in the presence of 18% salt is as low as 50% relative to 100% activity in the absence of salt, so there is the disadvantage of its inability to act adequately in the presence of high conc. salt. Similarly, the activity of the latter in the presence of 18% salt is also as low as 70% and is thus not satisfactory.
The former and latter glutaminases are thermostable up to 60.degree. C. respectively and are not satisfactory as compared with the thermostability of other kind of enzymes, so the advent of glutaminase further superior in thermostability is desired.
Accordingly, the object of the present invention is to obtain a microorganism having the ability to produce glutaminase significantly excellent in salt resistance and thermostability capable of acting adequately even in treatment at high temperatures in the presence of high conc. salt, and to produce salt-resistant thermostable glutaminase by use of this microorganism, as well as to acquire glutamic acid-rich protein hydrolysates (e.g. soy sauce) efficiently in a simple operation.